AQPX-cluster aquaporins and aquaglyceroporins are asymmetrically distributed in trypanosomes.

Major Intrinsic Proteins (MIPs) are membrane channels that permeate water and other small solutes. Some trypanosomatid MIPs mediate the uptake of antiparasitic compounds, placing them as potential drug targets. However, a thorough study of the diversity of these channels is still missing. Here we place trypanosomatid channels in the sequence-function space of the large MIP superfamily through a sequence similarity network. This analysis exposes that trypanosomatid aquaporins integrate a distant cluster from the currently defined MIP families, here named aquaporin X (AQPX). Our phylogenetic analyses reveal that trypanosomatid MIPs distribute exclusively between aquaglyceroporin (GLP) and AQPX, being the AQPX family expanded in the Metakinetoplastina common ancestor before the origin of the parasitic order Trypanosomatida. Synteny analysis shows how African trypanosomes specifically lost AQPXs, whereas American trypanosomes specifically lost GLPs. AQPXs diverge from already described MIPs on crucial residues. Together, our results expose the diversity of trypanosomatid MIPs and will aid further functional, structural, and physiological research needed to face the potentiality of the AQPXs as gateways for trypanocidal drugs.

TryTransDB: A web-based resource for transport proteins in Trypanosomatidae.

TryTransDB is a web-based resource that stores transport protein data which can be retrieved using a standalone BLAST tool. We have attempted to create an integrated database that can be a one-stop shop for the researchers working with transport proteins of Trypanosomatidae family. TryTransDB (Trypanosomatidae Transport Protein Database) is a web based comprehensive resource that can fire a BLAST search against most of the transport protein sequences (protein and nucleotide) from Trypanosomatidae family organisms. This web resource further allows to compute a phylogenetic tree by performing multiple sequence alignment (MSA) using CLUSTALW suite embedded in it. Also, cross-linking to other databases helps in gathering more information for a certain transport protein in a single website.

Polyamine-Based Thiols in Trypanosomatids: Evolution, Protein Structural Adaptations, and Biological Functions.

SIGNIFICANCE: Major pathogenic enterobacteria and protozoan parasites from the phylum Euglenozoa, such as trypanosomatids, are endowed with glutathione (GSH)-spermidine (Sp) derivatives that play important roles in signaling and metal and thiol-redox homeostasis. For some Euglenozoa lineages, the GSH-Sp conjugates represent the main redox cosubstrates around which entire new redox systems have evolved. Several proteins underwent molecular adaptations to synthesize and utilize the new polyamine-based thiols. Recent Advances: The genomes of closely related organisms have recently been sequenced, which allows mining and analysis of gene sequences that belong to these peculiar redox systems. Similarly, the three-dimensional structures of several of these proteins have been solved, which allows for comparison with their counterparts in classical redox systems that rely on GSH/glutaredoxin and thioredoxin. CRITICAL ISSUES: The evolutionary and structural aspects related to the emergence and use of GSH-Sp conjugates in Euglenozoa are reviewed focusing on unique structural specializations that proteins developed to use N1,N8-bisglutathionylspermidine (trypanothione) as redox cosubstrate. An updated overview on the biochemical and biological significance of the major enzymatic activities is also provided. FUTURE DIRECTIONS: A thiol-redox system strictly dependent on trypanothione is a feature unique to trypanosomatids. The physicochemical properties of the polyamine-GSH conjugates were a major driving force for structural adaptation of proteins that use these thiols as ligand and redox cofactor. In fact, the structural differences of indispensable components of this system can be exploited toward selective drug development. Future research should clarify whether additional cellular processes are regulated by the trypanothione system. Antioxid. Redox Signal. 28, 463-486.

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins.

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by 3H- palmitic acid and phosphate (32Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC-MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.

Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms.

Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.

RNA folding pathways and kinetics using 2D energy landscapes.

RNA folding pathways play an important role in various biological processes, such as (i) the hok/sok (host-killing/suppression of killing) system in E. coli to check for sufficient plasmid copy number, (ii) the conformational switch in spliced leader (SL) RNA from Leptomonas collosoma, which controls trans splicing of a portion of the '5 exon, and (iii) riboswitches--portions of the 5' untranslated region of messenger RNA that regulate genes by allostery. Since RNA folding pathways are determined by the energy landscape, we describe a novel algorithm, FFTbor2D, which computes the 2D projection of the energy landscape for a given RNA sequence. Given two metastable secondary structures A, B for a given RNA sequence, FFTbor2D computes the Boltzmann probability p(x, y) = Z(x,y)/Z that a secondary structure has base pair distance x from A and distance y from B. Using polynomial interpolationwith the fast Fourier transform,we compute p(x, y) in O(n(5)) time and O(n(2)) space, which is an improvement over an earlier method, which runs in O(n(7)) time and O(n(4)) space. FFTbor2D has potential applications in synthetic biology, where one might wish to design bistable switches having target metastable structures A, B with favorable pathway kinetics. By inverting the transition probability matrix determined from FFTbor2D output, we show that L. collosoma spliced leader RNA has larger mean first passage time from A to B on the 2D energy landscape, than 97.145% of 20,000 sequences, each having metastable structures A, B. Source code and binaries are freely available for download at http://bioinformatics.bc.edu/clotelab/FFTbor2D. The program FFTbor2D is implemented in C++, with optional OpenMP parallelization primitives.

Insights into the molecular evolution of HslU ATPase through biochemical and mutational analyses.

The ATP-dependent HslVU complexes are found in all three biological kingdoms. A single HslV protease exists in each species of prokaryotes, archaea, and eukaryotes, but two HslUs (HslU1 and HslU2) are present in the mitochondria of eukaryotes. Previously, a tyrosine residue at the C-terminal tail of HslU2 has been identified as a key determinant of HslV activation in Trypanosoma brucei and a phenylalanine at the equivalent position to E. coli HslU is found in T. brucei HslU1. Unexpectedly, we found that an F441Y mutation in HslU enhanced the peptidase and caseinolytic activity of HslV in E. coli but it showed partially reduced ATPase and SulA degradation activity. Previously, only the C-terminal tail of HslU has been the focus of HslV activation studies. However, the Pro315 residue interacting with Phe441 in free HslU has also been found to be critical for HslV activation. Hence, our current biochemical analyses explore the importance of the loop region just before Pro315 for HslVU complex functionality. The proline and phenylalanine pair in prokaryotic HslU was replaced with the threonine and tyrosine pair from the functional eukaryotic HslU2. Sequence comparisons between multiple HslUs from three different biological kingdoms in combination with biochemical analysis of E. coli mutants have uncovered important new insights into the molecular evolutionary pathway of HslU.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

New insights about cross-reactive epitopes of six trypanosomatid genera revealed that Crithidia and Leptomonas have antigenic similarity to L. (L.) chagasi.

We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as Trypanosoma cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.

Abstract folding space analysis based on helices.

RNA has many pivotal functions especially in the regulation of gene expression by ncRNAs. Identification of their structure is an important requirement for understanding their function. Structure prediction alone is often insufficient for this task, due to algorithmic problems, parameter inaccuracies, and biological peculiarities. Among the latter, there are base modifications, cotranscriptional folding leading to folding traps, and conformational switching as in the case of riboswitches. All these require more in-depth analysis of the folding space. The major drawback, which all methods have to cope with, is the exponential growth of the folding space. Therefore, methods are often limited in the sequence length they can analyze, or they make use of heuristics, sampling, or abstraction. Our approach adopts the abstraction strategy and remedies some problems of existing methods. We introduce a position-specific abstraction based on helices that we term helix index shapes, or hishapes for short. Utilizing a dynamic programming framework, we have implemented this abstraction in the program RNAHeliCes. Furthermore, we developed two hishape-based methods, one for energy barrier estimation, called HiPath, and one for abstract structure comparison, termed HiTed. We demonstrate the superior performance of HiPath compared to other existing methods and the competitive accuracy of HiTed. RNAHeliCes, together with HiPath and HiTed, are available for download at http://www.cyanolab.de/software/RNAHeliCes.htm.

Euglena gracilis and Trypanosomatids possess common patterns in predicted mitochondrial targeting presequences.

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic α-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5-118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.

Identification, molecular and functional characterization of calmodulin gene of Phytomonas serpens 15T that shares high similarity with its pathogenic counterparts Trypanosoma cruzi.

In trypanosomatids, Ca²+-binding proteins can affect parasite growth, differentiation and invasion. Due to their importance for parasite maintenance, they become an attractive target for drug discovery and design. Phytomonas serpens 15T is a non-human pathogenic trypanosomatid that expresses important protein homologs of human pathogenic trypanosomatids. In this study, the coding sequence of calmodulin, a Ca²+-binding protein, of P. serpens 15T was cloned and characterized. The encoded polypeptide (CaMP) displayed high amino acid identity to homolog protein of Trypanosoma cruzi and four helix-loop-helix motifs were found. CaMP sequence analysis showed 20 amino acid substitutions compared to its mammalian counterparts. This gene is located on a chromosomal band with estimated size of 1,300 kb and two transcripts were detected by Northern blot analysis. A polyclonal antiserum raised against the recombinant protein recognized a polypeptide with an estimated size of 17 kDa in log-phase promastigote extracts. The recombinant CaMP retains its Ca²+-binding capacity.

Mitochondrial redox metabolism in trypanosomatids is independent of tryparedoxin activity.

Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfur-containing peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently.

Polyamine biosynthesis in Phytomonas: biochemical characterisation of a very unstable ornithine decarboxylase.

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5'-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.

Leishmanolysin-like molecules in Herpetomonas samuelpessoai mediate hydrolysis of protein substrates and interaction with insect.

Herpetomonas samuelpessoai, an insect trypanosomatid, produces a 63-kDa metallopeptidase that has similar biochemical/immunological properties to Leishmania leishmanolysin, a virulence factor that participates in different stages of the parasite life cycle. Herein, we described some biochemical characteristics of the major surface metallopeptidase of H. samuelpessoai that led us to infer some probable functions for this peptidase during the parasite-invertebrate interaction. Gelatin-SDS-PAGE, flow cytometry and confocal fluorescence microscopy provided measurements for the relative levels of surface leishmanolysin-like molecules in H. samuelpessoai. Immunocytochemical analysis demonstrated the presence of leishmanolysin-like molecules on the surface and cytoplasm of the parasite. The surface metallopeptidase was active at a broad spectrum of pH and temperature, showing maximum activity at pH 6.0 at 37 degrees C, and an ability to degrade albumin, hemoglobin, IgG, mucin, casein and gut proteins obtained from Aedes aegypti. This wide substrate utilization might support parasite growth and development. Curiously, H. samuelpessoai cells were able to colonize A. aegypti guts. In an effort to implicate a possible role for the metallopeptidase from H. samuelpessoai, living parasites were treated with different compounds before the interaction with gut cells. The pre-incubation with metallopeptidase inhibitors, phospholipase C or anti-leishmanolysin antibodies promoted a significant reduction in the interaction with guts. Similarly, the pre-treatment of gut cells with purified leishmanolysin-like protein drastically diminished the adhesion process. Furthermore, the expression of surface leishmanolysin in H. samuelpessoai cells was drastically enhanced after passage in A. aegypti. These results suggest the participation of homologues of leishmanolysin in the interaction of H. samuelpessoai with the invertebrate vector.

Sialoglycoconjugates in Herpetomonas megaseliae: role in the adhesion to insect host epithelial cells.

Herpetomonas megaseliae is a monoxenic trypanosomatid isolated from the phorid fly Megaselia scalaris. In the present report, the expression of cell surface sialoglycoconjugates in this parasite was analyzed by Western blotting, flow cytometry and fluorescence microscopy analyses using lectins that specifically recognize sialic acid residues. A strong reaction was detected when parasites were treated with Limax flavus, Maackia amurensis and Sambucus nigra lectins. Analysis of crude protein extracts by Western blotting revealed that bands with molecular masses ranging from 19 to 80 kDa were reactive to these lectins, which showed a sugar-inhibited recognition with the parasite extract. These results indicated that molecules containing alpha2,3- and alpha2,6-sialylgalactosyl sequences are present in this protozoan. The role of the surface sialomolecules in the interaction with explanted guts from Aedes aegypti was assessed. The interaction of H. megaseliae with the insect gut was strongly inhibited in the presence of mucin (71%), fetuin (68%) and sialyllactose (68%). Collectively, our results suggest a possible involvement of sialomolecules in the interaction between this insect trypanosomatid and the invertebrate host.

Differential influence of gp63-like molecules in three distinct Leptomonas species on the adhesion to insect cells.

Parasites belonging to the Leptomonas genus have been used as model organisms for studying biochemical, cellular, and genetic processes unique to members of the Trypanosomatidae family. In the present study, the cell-associated and extracellular peptidases of three Leptomonas species, Leptomonas collosoma, Leptomonas samueli, and Leptomonas wallacei, were assayed and characterized by gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis. All parasites released metallopeptidases, whereas no cell-associated proteolytic activity could be detected in the cellular extracts from L. collosoma. Western blotting probed with a polyclonal antibody raised against gp63 from Leishmania amazonensis revealed two major reactive polypeptides of apparent molecular masses of 63 and 52 kDa, with different intensities in cellular extracts and released proteins from the studied trypanosomatids. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. This is the first report on the presence of gp63-like molecules in L. collosoma, L. samueli, and L. wallacei. The pretreatment of L. samueli and L. wallacei with anti-gp63 antibody significantly diminished their association index to Aedes albopictus cell line (C6/36), suggesting a potential involvement of the gp63-like molecules in the interaction process of these insect trypanosomatids with the vector.

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m3G antibodies.

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

Telomere biology of trypanosomatids: beginning to answer some questions.

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Characterization of glycosomal RING finger proteins of trypanosomatids.

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.